Optical control of protein activity and localization by fusion to photochromic protein domains

ABSTRACT

Engineered fusion proteins comprising photochromic protein domains are disclosed. In particular, the inventors have constructed fusion proteins containing photoswitchable photochromic fluorescent protein domains linked to selected proteins and shown that such fusion proteins can be used to control the activity or localization of selected proteins with light.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 13/848,346, filed Mar. 21, 2013, which claims benefit benefit under35 U.S.C. §119(e) of provisional application Ser. No. 61/614,492, filedMar. 22, 2012, all of which applications are hereby incorporated byreference in their entireties.

TECHNICAL FIELD

The present invention pertains generally to the field of proteinengineering and methods of controlling the activity or cellularlocalization of proteins. In particular, the invention relates toengineered fusion proteins comprising photochromic protein domains andmethods of using them to control protein activity or localization withlight.

BACKGROUND

The ability to control protein localization and activity would beenormously beneficial for understanding and modulating protein functionin physiological processes. Several approaches have been developedpreviously for optical control of protein activity using naturalproteins and protein domains that change conformation upon lightabsorption, for example, using proteins such as rhodopsins,phytochromes, and cryptochromes, and LOV domains from phototropins andFKF1 (Airan et al. (2009) Nature 458:1025-1029; Inoue et al. (2005) Nat.Methods 2:415-418; Kennedy et al. (2010) Nat. Methods 7:973-975;Levskaya et al. (2009) Nature 461:997-1001; Szobota et al. (2007) Neuron54:535-545; Wu et al. (2009) Nature 461:104-108; and Yazawa et al.(2009) Nat. Biotechnol. 27:941-945). However, widespread implementationof these methods has been hindered by various problems, including thelimited applicability of the methods to only specific signaling pathways(Airan et al., supra), the need for exogenous cofactors (Levskaya etal., supra), slow kinetics of induction (Yazawa et al., supra),undesirable light-independent dimerization (Kennedy et al., supra), orthe toxicity of light at blue wavelengths (Szobota et al., supra; Wu etal., supra; Yazawa et al., supra). Furthermore, of all these strategies,only fusion to LOV domains has been used to control the activity of asingle protein, but this method generally requires extensivecustomization (Wu et al., supra; Strickland et al. (2008) Proc. Natl.Acad. Sci. U.S.A. 105:10709-10714; Strickland et al. (2010) Nat. Methods7:623-626; and Wu et al. (2011) Methods Enzymol. 497:393-407). Inaddition, none of these light-absorbing domains are capable ofcontrolling both protein localization by intermolecular interactions andfunction of a single polypeptide chain.

Thus, there remains a need for a simple to use system for controllingprotein localization and activity with light, which can be readilyapplied to a wide range of proteins.

SUMMARY

The invention relates to engineered fusion proteins comprisingphotochromic protein domains. In particular, the inventors haveconstructed fusion proteins containing a photoswitchable photochromicfluorescent protein. The inventors have further shown that fusionproteins comprising one or more photochromic fluorescent protein domainslinked to a selected protein of interest can be used to control theactivity or localization of the selected protein using light.

In one aspect, the invention includes a fusion protein comprising atleast one photochromic polypeptide connected to a selected polypeptideof interest, wherein the oligomerization state of the photochromicpolypeptide is controllable with light. The photochromic polypeptide maybe a photochromic protein, or a variant or polypeptide fragment thereofhaving fluorescence characteristics, wherein the fluorescencecharacteristics of the fusion protein are dependent on theoligomerization state of the photochromic polypeptide. For example,photochromic proteins including, but not limited to Dronpa, Padron,rsTagRFP, and mApple, or a variant or polypeptide fragment thereofhaving fluorescence characteristics (e.g., Dronpa-145N, Padron-145N, ormApple-162H-164A), may be used in fusion constructs. In certainembodiments, the fusion protein comprises at least one photochromicpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 3, 5, 7, and 9 or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto.

In certain embodiments, the fusion protein comprises at least twophotochromic polypeptides, wherein a first photochromic polypeptide isconnected to the N-terminus of the selected polypeptide of interest anda second photochromic polypeptide is connected to the C-terminus of theselected polypeptide of interest, wherein the oligomerization state ofthe first photochromic polypeptide and the second photochromicpolypeptide is controllable with light. For example, the fusion proteinmay comprise two or more Dronpa, Padron, rsTagRFP, or mApplepolypeptides. The photochromic polypeptides in the fusion protein can bethe same or different.

In certain embodiments, the fusion protein comprises a Dronpa protein,or a variant or polypeptide fragment thereof having fluorescence andoligomerization characteristics. The fusion protein may comprise atleast one Dronpa 145N or Dronpa 145 K polypeptide. In certainembodiments, the fusion protein comprises two Dronpa polypeptides, whichcan be the same or different. For example, the fusion protein maycomprise two Dronpa 145N polypeptides, or two Dronpa 145K polypeptides,or a Dronpa 145K polypeptide and a Dronpa 145N polypeptide.

In certain embodiments, the fusion protein further comprises one or morelinkers connecting polypeptides within the fusion protein. Linkers aretypically short peptide sequences of 2-30 amino acid residues, oftencomposed of glycine and/or serine residues. Linker sequences that can beused in the practice of the invention include, but are not limited to[Gly]_(x), [Gly-Ser]_(x), [Gly-Gly-Ser-Gly]_(x), [Ser-Ala-Gly-Gly]_(x),and [Gly-Gly-Gly-Gly-Ser]_(x), wherein x=1-15, and GSAT, SEG, and Z-EGFRlinkers.

In certain embodiments, the fusion protein further comprises a targetingsequence. Targeting sequences that can be used in the practice of theinvention include, but are not limited to a secretory protein signalsequence, a membrane protein signal sequence, a nuclear localizationsequence, a nucleolar localization signal sequence, an endoplasmicreticulum localization sequence, a peroxisome localization sequence, amitochondrial localization sequence, and a protein-protein interactionmotif sequence.

In certain embodiments, the fusion protein further comprises a tag. Tagsthat can be used in the practice of the invention include, but are notlimited to a His-tag, a Strep-tag, a TAP-tag, an S-tag, an SBP-tag, anArg-tag, a calmodulin-binding peptide tag, a cellulose-binding domaintag, a DsbA tag, a c-myc tag, a glutathione S-transferase tag, a FLAGtag, a HAT-tag, a maltose-binding protein tag, a NusA tag, and athioredoxin tag.

The fusion proteins described herein can be used to control the activityor localization of a selected protein, which may be a membrane protein,a receptor, a hormone, a transport protein, a transcription factor, acytoskeletal protein, an extracellular matrix protein, asignal-transduction protein, an enzyme, or any other protein ofinterest. The fusion protein may comprise the entire protein, or abiologically active domain (e.g., a catalytic domain, a ligand bindingdomain, or a protein-protein interaction domain), or a polypeptidefragment of the selected protein of interest.

In another aspect, the invention includes a method for controlling theactivity of a selected polypeptide of interest with light. The methodcomprises (i) preparing a fusion protein comprising a first photochromicpolypeptide connected to the N-terminus of the selected polypeptide ofinterest and a second photochromic polypeptide connected to theC-terminus of the selected polypeptide of interest; (ii) illuminatingthe fusion protein with light at a wavelength that inducesintramolecular dimerization of the first photochromic polypeptide andthe second photochromic polypeptide (e.g., about 405 nm for some fusionswith Dronpa 145N or 145K), such that the activity of the selectedpolypeptide of interest is inactivated. In certain embodiments, themethod further comprises illuminating the fusion protein with light at awavelength that induces dissociation of the first photochromicpolypeptide from the second photochromic polypeptide (e.g., about480-500 nm for some fusions with Dronpa 145N or 145K), such that theactivity of the selected polypeptide is restored. Localization of theselected polypeptide as well as inactivation of the selected polypeptidecan be visualized by detecting fluorescence of the fusion proteinresulting from intramolecular dimerization of the first photochromicpolypeptide and the second photochromic polypeptide in the fusionprotein. Inactivation of the selected polypeptide can further beassessed by measuring the activity of the selected polypeptide.

In another aspect, the invention includes a method for controlling thelocalization of a selected polypeptide of interest with light. Themethod comprises (i) preparing a first fusion protein comprising aphotochromic polypeptide connected to a targeting sequence; (ii)preparing a second fusion protein comprising a photochromic polypeptideconnected to the selected polypeptide of interest; (iii) introducing thefirst fusion protein and the second fusion protein into a cell, whereinthe localization sequence targets the first fusion protein to aparticular subcellular location; (iv) and illuminating the fusionproteins with light at a wavelength that induces oligomerization of thephotochromic polypeptide in the first fusion protein with thephotochromic polypeptide in the second fusion protein (e.g., about 405nm for some fusions with Dronpa 145N or 145K), such that the selectedpolypeptide of interest accumulates at the subcellular location. Incertain embodiments, the method further comprises illuminating thefusion proteins with light at a wavelength that induces dissociation ofthe photochromic polypeptides (e.g., about 480-500 nm for some fusionswith Dronpa 145N or 145K), such that the selected polypeptide in thesecond fusion protein is released from the subcellular location.Localization of the selected polypeptide can be visualized by detectingfluorescence of the fusion proteins resulting from the oligomerizationof the photochromic polypeptides.

In another aspect, the invention includes a method for controlling thelocalization of a selected polypeptide of interest with light. Themethod comprises: (i) preparing a fusion protein comprising aphotochromic polypeptide, a targeting sequence, and the selectedpolypeptide of interest; (ii) introducing the fusion protein into acell, wherein the localization sequence targets the fusion protein to aparticular subcellular location; and (iii) illuminating the fusionprotein with light at a wavelength that induces oligomerization of thephotochromic polypeptide in the fusion protein with photochromicpolypeptides in other fusion proteins (e.g., 405 nm for some fusionswith Dronpa 145N or 145K), the other fusion proteins comprising theselected polypeptide, such that the selected polypeptide accumulates atthe subcellular location. In certain embodiments, the method furthercomprises illuminating the fusion protein with light at a wavelengththat induces dissociation of the photochromic polypeptides (e.g, about480-500 nm for some fusions with Dronpa 145N or 145K), such that theselected polypeptide in the fusion protein is released from thesubcellular location. Localization of the selected polypeptide can bevisualized by detecting fluorescence of the fusion protein resultingfrom the oligomerization with photochromic polypeptides of the otherfusion proteins.

In another aspect, the invention includes a polynucleotide encoding afusion protein described herein. In one embodiment, the polynucleotideis a recombinant polynucleotide comprising a polynucleotide encoding afusion protein operably linked to a promoter. In certain embodiments,the recombinant polynucleotide comprises a polynucleotide selected fromthe group consisting of: a polynucleotide encoding a polypeptidecomprising a sequence selected from the group consisting of SEQ IDNOS:1, 3, 5, 7, and 9; a polynucleotide encoding a polypeptidecomprising a sequence having at least 95% identity to a sequenceselected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9; apolynucleotide comprising a sequence selected from the group consistingof SEQ ID NOS:2, 4, 6, 8, and 10; and a polynucleotide comprising asequence having at least 95% identity to a sequence selected from thegroup consisting of SEQ ID NOS:2, 4, 6, 8, and 10.

In another aspect, the invention includes a host cell comprising arecombinant polynucleotide encoding a fusion protein operably linked toa promoter.

In another aspect, the invention includes a method for producing afusion protein, the method comprising: transforming a host cell with arecombinant polynucleotide encoding a fusion protein operably linked toa promoter; culturing the transformed host cell under conditions wherebythe fusion protein is expressed; and isolating the fusion protein fromthe host cell.

In another aspect, the invention includes a kit for preparing or usingfusion proteins according to the methods described herein. Such kits maycomprise one or more photochromic polypeptides or fusion proteins, ornucleic acids encoding such polypeptides or fusion proteins, orexpression vectors, or cells, or other reagents for preparingpolypeptides and fusion proteins, as described herein.

In the practice of the invention, the fluorescence of fusion proteinscan be monitored by any suitable method. For example, fluorescence offusion proteins can be detected by a fluorometer, a fluorescencemicroscope, a fluorescence microplate reader, a fluorometric imagingplate reader, or fluorescence-activated cell sorting.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F show the control of photochromic fluorescent protein (FP)domain association by light. FIG. 1A shows a schematic representation ofthe hypothesized bidirectional control of the Dronpa145N oligomerizationstate by 500-nm cyan and 400-nm violet light. FIG. 1B shows nativepolyacrylamide gel electrophoresis (PAGE) of Dronpa145N (100 μM), whichdemonstrated that 500 nm light induced dissociation and 400 nm lightinduced retetramerization. The mRuby2 (Lam et al. (2012) Nat. Methods9:1005-1012), tdTomato, and dsRed2 (20 μM) served as monomeric, dimeric,and tetrameric standards, respectively. All proteins werepolyhistidine-tagged at the amino terminus (NT). FIG. 1C showsabsorbance spectra confirming that photoswitching is reversible. FIG. 1Dshows a schematic representation of the hypothesized bidirectionalconformational switching by light in a Dronpa145K-Dronpa145N (K-N)tandem dimer. FIG. 1E shows that native PAGE of the K-N tandem dimerdemonstrated faster migration by the K-N tandem dimer (100 μM) afterexposure to 500-nm light, an effect that was reversed by 400-nm light.The asterisk marks the location expected for tandem dimer migration,similar to tdTomato. Some cleavage of the tandem dimer to a monomer inthis protein preparation was apparent. FIG. 1F shows absorbance spectraof K-N tandem dimers confirming that photoswitching is reversible.

FIGS. 2A-2H show the control of photochromic FP domain association bylight in cells. FIG. 2A shows the experimental plan for light-regulatedinteraction between Dronpa145N-CAAX (N-CAAX) and mNeptune-Dronpa145N(mNeptune-N). FIG. 2B shows quantitation of membrane Dronpa fluorescenceduring 490/20-nm illumination. FIG. 2C shows that 490/20-nm lightinduced off-photoswitching of Dronpa and loss of mNeptune from theplasma membrane (scale bar, 20 μm). FIG. 2D shows an intensity profilefor the region between the arrows shown in FIG. 2C. FIG. 2E shows theexperimental plan for light-regulated interaction betweenDronpa145K-CAAX (K-CAAX) and mNeptune-N. FIG. 2F shows the quantitationof membrane Dronpa fluorescence during 490/20-nm illumination. FIG. 2Gshows that 490/20-nm light induced off-photoswitching of Dronpa and lossof mNeptune from the membrane. mNeptune reappeared at the membrane after3-seconds of on-photoswitching with 390/15-nm light (scale bar, 20 μm).FIG. 2H shows intensity profiles for the region between the arrows shownin FIG. 2G.

FIGS. 3A-3H show a light-inducible single-chain guanine nucleotideexchange factor (GEF). FIG. 3A shows the proposed mechanism forphoto-uncaging of N-I-N-CAAX activity (construct contained Dronpa145N atthe N-terminus of the intersectin (ITSN) Dbl homology (DH) domain andDronpa145N at the C-terminus followed by the CAAX sequence). FIG. 3Bshows off-photoswitching of Dronpa fluorescence in N-I-N-CAAX versus490/20-nm light dosage during microscopy. Whole-cell fluorescenceresults from five cells were quantified and normalized to the initialvalue. Error bars represent standard deviation (SD). FIG. 3C shows thatin NIH 3T3 cells expressing N-I-N-CAAX, 490/20-nm illumination for 30seconds (off-switching) followed by incubation at 37° C. for 30 minutesresulted in robust induction of filopodia, as revealed bymNeptune-Fascin. FIG. 3D shows that local illumination by 490/20-nmlight locally induced filopodia, marked by mNeptune-fascin, in NIH 3T3cells expressing N-I-N-CAAX. The dotted curves indicate the area ofillumination. FIG. 3E shows the proposed mechanism for photo-uncaging ofK-I-N-CAAX activity (construct contained Dronpa145K at the N-terminus ofthe intersectin (ITSN) Dbl homology (DH) domain and Dronpa145N at theC-terminus followed by the CAAX sequence). FIG. 3F showsoff-photoswitching of Dronpa fluorescence in K-I-N-CAAX versus 490/20-nmlight dosage during microscopy. The experiment was performed asdescribed for FIG. 3B. FIG. 3G shows that in NIH 3T3 cells expressingK-I-N-CAAX, exposure to 490/20-nm light for 30 seconds (off-switching)followed by incubation at 37° C. for 30 minutes resulted in robustinduction of filopodia. FIG. 3H shows that local illumination by490/20-nm light locally induced filopodia, marked by mNeptune-fascin, inNIH 3T3 cells expressing K-I-N-CAAX. The dotted curves indicate the areaof illumination. The scale bars in FIGS. 3C, 3D, 3G, and 3H are 20 μm.

FIGS. 4A-4C show results with a light-inducible single-chain protease,N-protease-N (Dronpa145N-protease-Dronpa145N fusion). FIG. 4A shows thestrategy for sensing activity of the N-protease-N protein withmCherry-substrate-CAAX. FIG. 4B shows the distribution of mCherry incells expressing mCherry-substrateCAAX in the absence (left) or presence(middle) of cotransfected K-protease. The chart at right shows thefluorescence intensity profile along the line between the arrows in theimages. FIG. 4C shows that as expected from its size (81 kD),N-protease-N was excluded from the nucleus (left). Exposure to 490/20-nmlight for 15 seconds induced off-photoswitching of Dronpa fluorescence(Dronpa channel) and induced release of mCherry from the membrane(mCherry channel). The chart at right shows the intensity profile alongthe line between the arrows in the images, which confirmed that mCherryfluorescence decreases from the membrane and increases in the cytosoland nucleus after illumination. The scale bars in FIGS. 4B and 4C are 20μm.

FIGS. 5A-5C show quantification of reversible photoswitching ofrecombinant Dronpa constructs by fluorescence in vitro. FIG. 5A shows anative PAGE, which demonstrated that Dronpa145K is purely monomeric and145N is predominantly tetrameric at concentrations from 10 μM to 100 μM.The mRuby2, tdTomato, and dsRed2 served as monomeric, dimeric, andtetrameric standards, respectively. FIG. 5B shows that fluorescence ofDronpa145N was switched off by 500 nm light and switched back on by 400nm light using the same conditions as described for FIG. 1. Fluorescencewas measured in quadruplicate at 480/5 nm excitation and 530/5 nmemission and intensities normalized to the initial value. Error barsrepresent standard deviation. FIG. 5C shows that fluorescence of theDronpa145K-Dronpa145N tandem dimer was switched off by 500 nm light andswitched back on by 400 nm light using the same conditions as describedfor FIG. 1.

FIGS. 6A and 6B show the directional specificity of membrane recruitmentof Dronpa. FIG. 6A shows that N-CAAX did not recruit mNeptune-K to themembrane, possibly due to N—N intramembrane homotetramerizationoutcompeting K-N heterodimerization. FIG. 6B shows that K-CAAX wasunable to recruit mNeptune-K to the membrane, confirming mNeptunemembrane localization required Dronpa multimerization. Scale bars are 10μm.

FIGS. 7A-7D show protein caging by fusion to interacting FP domains.FIG. 7A shows structural models of a DH domain caged by two flankingDronpa145N domains (top), or flanking Dronpa145K and Dronpa145N domains(bottom). FIG. 7B shows the organization of the control intersectinDbl-homology domain (ITSN DH) constructs, caged ITSN DH constructs, anda mNeptune-fascin filopodia reporter. FIG. 7C shows representative NIH3T3 fibroblasts expressing K-CAAX, K-I-CAAX, I-K-CAAX, N-I-N-CAAX, orK-I-N-CAAX (Dronpa channel) with filopodia and lamellipodia marked bymNeptune-fascin (mNeptune channel). The scale bar is 10 μm. FIG. 7Dshows the frequency of filopodia or lamellipodia formation in cellstransiently transfected with various constructs. Cells showinglamellipodia or more than one filopodium at one polygonal side werescored as positive. Numbers above the bars are the number of cells ineach condition. All imaged cells were scored. The scale bars are 10 μm.

FIGS. 8A-8D show the quantitation of filopodia and lamellipodiaproduction by Dronpa-intersectin fusion constructs at differentexpression levels. FIG. 8A shows the distribution of Dronpa fluorescenceintensities from 37 cells transfected with N-I-N-CAAX. Boundaries fordefining low, medium, and high expressers are shown as dotted lines.FIG. 8B shows the occurrence of filopodia or lamellipodia is low incells expressing low levels of N-I-N-CAAX. FIG. 8C shows thedistribution of Dronpa fluorescence intensities from 50 cellstransfected with K-I-N-CAAX. Boundaries for defining low, medium, andhigh expressers are shown as dotted lines. FIG. 8D shows that theoccurrence of filopodia or lamellipodia is low in cells expressing lowlevels of K-I-N-CAAX.

FIGS. 9A-9D show that filopodia induction required both light and acaged ITSN DH protein. FIG. 9A shows that cells expressing N-I-N-CAAXdid not produce filopodia without illumination. Initial Dronpa was notimaged to avoid uncaging by the 500 nm excitation light. FIG. 9B showsthat cells expressing K-I-N-CAAX did not produce filopodia underidentical conditions without illumination. Initial Dronpa was not imagedto avoid uncaging by the 490/20 nm excitation light. FIG. 9C shows thatin cells expressing K-CAAX (lacking ITSN DH), 490/20 nm illumination for30 seconds (Dronpa off-switching) followed by incubation at 37° C. for30 minutes did not produce filopodia or lamellipodia. FIG. 9D shows thatquantitation of new filopodia and lamellipodia formation with lightstimulation in cells expressing N-I-N-CAAX and K-I-N-CAAX. Numbers abovethe bars are the number of cells in each condition. All imaged cellswere scored. The scale bars are 10 μm.

FIG. 10 shows temporal regulation of filopodia by light induction ofintersectin activity. In a NIH3T3 cell expressing N-I-N-CAAX, localuncaging with 500 nm light (frame 1, dotted circle) induced localfilopodia formation in 10 minutes (arrow, frames 2-3). The cell was thenglobally illuminated with 400 nm light to recage N-I-N-CAAX, then localuncaging was performed in a new location with 500 nm light (frame 4,dotted circle). Filopodia in the first uncaging location subsequentlyretracted (asterisk) while new filopodia formed in the second uncaginglocation (arrows, frames 5-7). The scale bar is 20 μm.

FIG. 11 shows that optical induction of intersectin reveals a role forCdc42 in filopodia elongation. Uncaging of K-I-N-CAAX in a cellexpressing preexisting filopodia (arrows) results in lengthening of thefilopodia. New filopodia formation can also be observed (asterisk). Thescale bar is 20 μm.

FIGS. 12A and 12B show that release of mCherry from themCherry-substrate-CAAX fusion required both light and the cagedprotease. FIG. 12A shows that HEK293 cells expressingmCherry-substrate-CAAX together with N-protease-N did not releasemCherry from the membrane in the absence of light stimulation. FIG. 12Bshows that cells expressing mCherry-substrate-CAAX alone did not releasemCherry from the membrane even after light stimulation. Scale bars are10 μm.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of pharmacology, chemistry,biochemistry, recombinant DNA techniques and immunology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., Blackwell Scientific Publications); A. L.Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al., Molecular Cloning: A Laboratory Manual (3^(rd)Edition, 2001); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

I. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a fusion protein” includes a mixture of two or more fusionproteins, and the like.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

“Fluorescent protein” refers to any protein capable of emitting lightwhen excited with appropriate electromagnetic radiation. Fluorescentproteins include proteins having amino acid sequences that are eithernatural or engineered (e.g., Dronpa, Padron, rsTagRFP, and mApple, andvariants and derivatives thereof).

A Dronpa polynucleotide, nucleic acid, oligonucleotide, protein,polypeptide, or peptide refers to a molecule derived from a coral of thegenus Pectiniidae. The molecule need not be physically derived fromPectiniidae, but may be synthetically or recombinantly produced. Anumber of Dronpa nucleic acid and protein sequences are known.Representative Dronpa sequences are presented in SEQ ID NOS:1-4.Additional representative sequences are listed in the National Centerfor Biotechnology Information (NCBI) database. See, for example, NCBIentries: Accession Nos. AB180726, ADE48854, BAD72874.1, 2IOV_D, 2IOV_C,2IOV_B, 2IOV_A, 2POX_D, 2POX_C, 2POX_B, 2POX_A, AED56657, AED56658,AED56659, and AED56660; all of which sequences (as entered by the dateof filing of this application) are herein incorporated by reference. Anyof these sequences or a variant thereof comprising a sequence having atleast about 80-100% sequence identity thereto, including any percentidentity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto,can be used to construct a fusion protein, as described herein.

A Padron polynucleotide, nucleic acid, oligonucleotide, protein,polypeptide, or peptide refers to a molecule derived from Echinophylliasp. SC22. The molecule need not be physically derived from Echinophylliasp., but may be synthetically or recombinantly produced. A number ofPadron nucleic acid and protein sequences are known. RepresentativePadron sequences are presented in SEQ ID NO:5 and SEQ ID NO:6.Additional representative sequences are listed in the National Centerfor Biotechnology Information (NCBI) database. See, for example, NCBIentries: Accession Nos. ACL36360, ACL98050, EU983551, FJ014613, 3ZUL_A,3ZUL_B, 3ZUL_C, 3ZUL_D, 3ZUL_E, 3ZUL_F, 3ZUJ_A, 3ZUJ_B, 3ZUJ_C, 3ZUJ_D,3ZUJ_E, 3ZUJ_F, 3ZUF_A, 3ZUF_B, 3ZUF_C, 3ZUF_D, 3ZUF_E, and 3ZUF_F; allof which sequences (as entered by the date of filing of thisapplication) are herein incorporated by reference. Any of thesesequences or a variant thereof comprising a sequence having at leastabout 80-100% sequence identity thereto, including any percent identitywithin this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can beused to construct a fusion protein, as described herein.

An rsTagRFP polynucleotide, nucleic acid, oligonucleotide, protein,polypeptide, or peptide refers to a molecule derived from Entacmaeaquadricolor. The molecule need not be physically derived from Entacmaeaquadricolor, but may be synthetically or recombinantly produced. Anumber of rsTagRFP nucleic acid and protein sequences are known.Representative rsTagRFP sequences are presented in SEQ ID NO:7 and SEQID NO:8. Additional representative sequences are listed in the NationalCenter for Biotechnology Information (NCBI) database. See, for example,NCBI entries: Accession Nos. 3U8C_A, 3U8C_B, 3U8C_C, 3U8C_D, 3U8A_A,3U8A_B, 3U8A_C, 3U8A_D; all of which sequences (as entered by the dateof filing of this application) are herein incorporated by reference. Anyof these sequences or a variant thereof comprising a sequence having atleast about 80-100% sequence identity thereto, including any percentidentity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto,can be used to construct a fusion protein, as described herein.

An mApple polynucleotide, nucleic acid, oligonucleotide, protein,polypeptide, or peptide refers to a molecule derived from Discosoma sp.The molecule need not be physically derived from Discosoma sp., but maybe synthetically or recombinantly produced. A number of mApple nucleicacid and protein sequences are known. Representative mApple sequencesare presented in SEQ ID NO:9 and SEQ ID NO:10. Additional representativesequences are listed in the National Center for BiotechnologyInformation (NCBI) database. See, for example, NCBI entries: AccessionNos. ABC66097, DQ336160; all of which sequences (as entered by the dateof filing of this application) are herein incorporated by reference. Anyof these sequences or a variant thereof comprising a sequence having atleast about 80-100% sequence identity thereto, including any percentidentity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto,can be used to construct a fusion protein, as described herein.

The terms “fusion protein,” “fusion polypeptide,” or “photochromicfusion protein” as used herein refer to a fusion comprising at least onephotochromic polypeptide in combination with a selected polypeptide ofinterest as part of a single continuous chain of amino acids, whichchain does not occur in nature. The photochromic polypeptides and otherselected polypeptides may be connected directly to each other by peptidebonds or may be separated by intervening amino acid sequences. Thefusion may include entire proteins or fragments thereof, including, forexample, sequences of Dronpa, Padron, rsTagRFP, mApple, or variantsthereof having fluorescence characteristics (e.g., Dronpa-145K,Dronpa-145N, Padron-145N, and mApple-162H-164A). The fusion polypeptidesmay also contain sequences exogenous to the photochromic or otherselected polypeptides. For example, the fusion may include targeting orlocalization sequences, tag sequences, sequences of other fluorescentproteins (e.g., other proteins with fluorescence characteristics thatdiffer from Dronpa, Padron, rsTagRFP, or mApple), or other chromophores.Moreover, the fusion may contain sequences from multiple photochromicproteins, or variants thereof, and/or other selected proteins. Forexample, the fusion protein may comprise two or more Dronpa, Padron,rsTagRFP, or mApple polypeptides, which can be the same or different(e.g., two or more Dronpa 145K or Dronpa 145N polypeptides, or a Dronpa145K polypeptide and a Dronpa 145N polypeptide simultaneously in thesame fusion). Alternatively, the fusion protein may comprise only onephotochromic polypeptide, which can be a wild-type polypeptide, orvariant thereof.

The term “fluorescence characteristics” means an ability to emitfluorescence by irradiation of excitation light. The fluorescencecharacteristics of a fluorescent fusion protein comprising aphotochromic polypeptide or a variant thereof may be comparable to ordifferent from those of the fluorescent proteins which have the aminoacid sequences shown in SEQ ID NOS:1, 3, 5, 7, and 9. Examples ofparameters of the fluorescence characteristics include fluorescenceintensity, excitation wavelength, fluorescence wavelength, and pHsensitivity.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length. Thus, peptides,oligopeptides, dimers, multimers, and the like, are included within thedefinition. Both full length proteins and fragments thereof areencompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation, hydroxylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions andsubstitutions to the native sequence, so long as the protein maintainsthe desired activity. These modifications may be deliberate, as throughsite directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

By “derivative” is intended any suitable modification of the nativepolypeptide of interest, of a fragment of the native polypeptide, or oftheir respective analogs, such as glycosylation, phosphorylation,polymer conjugation (such as with polyethylene glycol), or otheraddition of foreign moieties, as long as the desired biological activityof the native polypeptide is retained. Methods for making polypeptidefragments, analogs, and derivatives are generally available in the art.

By “fragment” is intended a molecule consisting of only a part of theintact full length sequence and structure. The fragment can include aC-terminal deletion an N-terminal deletion, and/or an internal deletionof the polypeptide. Active fragments of a particular protein orpolypeptide will generally include at least about 5-10 contiguous aminoacid residues of the full length molecule, preferably at least about15-25 contiguous amino acid residues of the full length molecule, andmost preferably at least about 20-50 or more contiguous amino acidresidues of the full length molecule, or any integer between 5 aminoacids and the full length sequence, provided that the fragment inquestion retains biological activity, such as catalytic activity, ligandbinding activity, regulatory activity, fluorescence or oligomerizationcharacteristics, as defined herein.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample, a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.The term “fluorescer” refers to a substance or a portion thereof whichis capable of exhibiting fluorescence in the detectable range. The termalso includes fluorescent proteins and polypeptides.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50% sequence identity, preferably at least about 75%sequence identity, more preferably at least about 80% 85% sequenceidentity, more preferably at least about 90% sequence identity, and mostpreferably at least about 95% 98% sequence identity over a definedlength of the molecules. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide to nucleotide oramino acid to amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353 358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482 489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single stranded specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation, is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

The term “transformation” refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction or f-mating areincluded. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

“Recombinant host cells”, “host cells,” “cells”, “cell lines,” “cellcultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence can bedetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic DNA sequences from viral or prokaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements,” include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. An expression cassette generally includes controlelements, as described above, such as a promoter which is operablylinked to (so as to direct transcription of) the sequence(s) or gene(s)of interest, and often includes a polyadenylation sequence as well.Within certain embodiments of the invention, the expression cassettedescribed herein may be contained within a plasmid construct. Inaddition to the components of the expression cassette, the plasmidconstruct may also include, one or more selectable markers, a signalwhich allows the plasmid construct to exist as single stranded DNA(e.g., a M13 origin of replication), at least one multiple cloning site,and a “mammalian” origin of replication (e.g., a SV40 or adenovirusorigin of replication).

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout at least 90%, of the protein with which the polynucleotide isnaturally associated. Techniques for purifying polynucleotides ofinterest are well-known in the art and include, for example, disruptionof the cell containing the polynucleotide with a chaotropic agent andseparation of the polynucleotide(s) and proteins by ion-exchangechromatography, affinity chromatography and sedimentation according todensity.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratorymanual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis etal. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill,and Chu et al. (1981) Gene 13:197. Such techniques can be used tointroduce one or more exogenous DNA moieties into suitable host cells.The term refers to both stable and transient uptake of the geneticmaterial, and includes uptake of peptide- or antibody-linked DNAs.

A “vector” is capable of transferring nucleic acid sequences to targetcells (e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a nucleic acid of interest and which cantransfer nucleic acid sequences to target cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule that retain desired activity, suchas fluorescence or oligomerization characteristics. In general, theterms “variant” and “analog” refer to compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy biological activity and which are “substantiallyhomologous” to the reference molecule as defined below. In general, theamino acid sequences of such analogs will have a high degree of sequencehomology to the reference sequence, e.g., amino acid sequence homologyof more than 50%, generally more than 60%-70%, even more particularly80%-85% or more, such as at least 90%-95% or more, when the twosequences are aligned. Often, the analogs will include the same numberof amino acids but will include substitutions, as explained herein. Theterm “mutein” further includes polypeptides having one or more aminoacid-like molecules including but not limited to compounds comprisingonly amino and/or imino molecules, polypeptides containing one or moreanalogs of an amino acid (including, for example, unnatural amino acids,etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring (e.g., synthetic), cyclized, branched moleculesand the like. The term also includes molecules comprising one or moreN-substituted glycine residues (a “peptoid”) and other synthetic aminoacids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005; 5,877,278; and5,977,301; Nguyen et al., Chem. Biol. (2000) 7:463-473; and Simon etal., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions ofpeptoids). Methods for making polypeptide analogs and muteins are knownin the art and are described further below.

As explained above, analogs generally include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, and tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA or RNA of interest into a host cell. Such methodscan result in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from bacterial plasmid vectors,viral vectors, non-viral vectors, alphaviruses, pox viruses and vacciniaviruses.

The term “derived from” is used herein to identify the original sourceof a molecule but is not meant to limit the method by which the moleculeis made which can be, for example, by chemical synthesis or recombinantmeans.

A polynucleotide “derived from” a designated sequence refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding, i.e.,identical or complementary to, a region of the designated nucleotidesequence. The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide.

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention is based on the discovery of engineered fusionproteins comprising photochromic fluorescent protein domains that can beused to control the activity or localization of a selected protein ofinterest. In particular, the inventors have constructed fusion proteinscontaining photoswitchable variants of the fluorescent protein Dronpa(see Example 1). Dronpa undergoes light-inducible oligomerization, whichconverts Dronpa from a dark form to a bright form with detectablefluorescence. Thus, fusions of Dronpa with a selected protein allow theprotein to be detected when Dronpa is converted to its bright form. Theinventors have further shown that fusion proteins comprising Dronpalinked to a selected protein of interest can be used to control theactivity or localization of the selected protein with light (see Example1). In order to further an understanding of the invention, a moredetailed discussion is provided below regarding photochromic fusionproteins and methods of using them to control the activity andlocalization of proteins.

A. Fusion Proteins

Fusion proteins comprise at least one photochromic polypeptide connectedto a selected polypeptide of interest. The fusion protein can bedesigned to block or induce activity of the selected polypeptide ofinterest, control its interactions with other macromolecules, or directits subcellular localization. The polypeptide of interest selected forstudy may be from a membrane protein, a receptor, a hormone, a transportprotein, a transcription factor, a cytoskeletal protein, anextracellular matrix protein, a signal-transduction protein, an enzyme,or any other protein of interest. The fusion protein may include entirephotochromic proteins, or biologically active domains or polypeptidefragments, or variants thereof having fluorescence characteristics(e.g., Dronpa-145K, Dronpa-145N, Padron-145N, rsTagRFP, andmApple-162H-164A). In addition, the fusion protein may comprise anentire selected protein of interest, or a biologically active domain(e.g., a catalytic domain, a ligand binding domain, or a protein-proteininteraction domain), or a polypeptide fragment of the selected proteinof interest.

Dronpa nucleic acid and protein sequences may be derived from corals ofthe genus Pectiniidae. A number of Dronpa nucleic acid and proteinsequences are known. Representative Dronpa sequences are presented inSEQ ID NOS:1-4 and additional representative sequences are listed in theNational Center for Biotechnology Information (NCBI) database. See, forexample, NCBI entries: Accession Nos. AB180726, ADE48854, BAD72874.1,2IOV_D, 2IOV_C, 2IOV_B, 2IOV_A, 2POX_D, 2POX_C, 2POX_B, 2POX_A,AED56657, AED56658, AED56659, and AED56660; all of which sequences (asentered by the date of filing of this application) are hereinincorporated by reference. Any of these sequences or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto, can be used to construct a fusionprotein, as described herein.

Padron nucleic acid and protein sequences may be derived fromEchinophyllia sp. SC22. A number of Padron nucleic acid and proteinsequences are known. Representative Padron sequences are presented inSEQ ID NO:5 and SEQ ID NO:6. Additional representative sequences arelisted in the National Center for Biotechnology Information (NCBI)database. See, for example, NCBI entries: Accession Nos. ACL36360,ACL98050, EU983551, FJ014613, 3ZUL_A, 3ZUL_B, 3ZUL_C, 3ZUL_D, 3ZUL_E,3ZUL_F, 3ZUJ_A, 3ZUJ_B, 3ZUJ_C, 3ZUJ_D, 3ZUJ_E, 3ZUJ_F, 3ZUF_A, 3ZUF_B,3ZUF_C, 3ZUF_D, 3ZUF_E, and 3ZUF_F; all of which sequences (as enteredby the date of filing of this application) are herein incorporated byreference. Any of these sequences or a variant thereof comprising asequence having at least about 80-100% sequence identity thereto,including any percent identity within this range, such as 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%sequence identity thereto, can be used to construct a fusion protein, asdescribed herein.

RsTagRFP nucleic acid and protein sequences may be derived fromDiscosoma sp. A number of rsTagRFP nucleic acid and protein sequencesare known. Representative rsTagRFP sequences are presented in SEQ IDNO:7 and SEQ ID NO:8. Additional representative sequences are listed inthe National Center for Biotechnology Information (NCBI) database. See,for example, NCBI entries: Accession Nos. 3U8C_A, 3U8C_B, 3U8C_C,3U8C_D, 3U8A_A, 3U8A_B, 3U8A_C, 3U8A_D; all of which sequences (asentered by the date of filing of this application) are hereinincorporated by reference. Any of these sequences or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto, can be used to construct a fusionprotein, as described herein.

MApple nucleic acid and protein sequences may be derived from Discosomasp. A number of mApple nucleic acid and protein sequences are known.Representative mApple sequences are presented in SEQ ID NO:9 and SEQ IDNO:10. Additional representative sequences are listed in the NationalCenter for Biotechnology Information (NCBI) database. See, for example,NCBI entries: Accession Nos. ABC66097, DQ336160; all of which sequences(as entered by the date of filing of this application) are hereinincorporated by reference. Any of these sequences or a variant thereofcomprising a sequence having at least about 80-100% sequence identitythereto, including any percent identity within this range, such as 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% sequence identity thereto, can be used to construct a fusionprotein, as described herein.

The photochromic polypeptides and other polypeptides included in thefusion construct may be connected directly to each other by peptidebonds or may be separated by intervening amino acid sequences. Thefusion polypeptides may also contain sequences exogenous to thephotochromic polypeptides or the selected protein of interest. Forexample, the fusion may include targeting or localization sequences, tagsequences, sequences of other fluorescent proteins (e.g., withfluorescence characteristics that differ from other photochromicproteins in the fusion protein), or other chromophores. Moreover, thefusion may contain sequences from multiple photochromic proteins, orvariants thereof, and/or non-photochromic proteins. For example, thefusion protein may comprise two or more photochromic polypeptides, whichcan be the same or different (e.g., two or more Dronpa 145K or Dronpa145N polypeptides, or a Dronpa 145K polypeptide and a Dronpa 145Npolypeptide simultaneously in the same fusion). Alternatively, thefusion protein may comprise only one photochromic polypeptide, which canbe a wild-type photochromic polypeptide, or a variant thereof.

In certain embodiments, the fusion protein can be represented by theformula NH₂-A-D-L-X-B-COOH or NH₂-A-X-L-D-B-COOH, wherein: D is an aminoacid sequence of a photochromic protein or a variant or polypeptidefragment thereof; L is an optional linker amino acid sequence; X is anamino acid sequence of a selected polypeptide of interest; A is anoptional N-terminal amino acid sequence; and B is an optional C-terminalamino acid sequence.

In other embodiments, the fusion protein can be represented by theformula NH₂-A-D₁-L-X-L-D₂-B-COOH, wherein: D₁ and D₂ are amino acidsequences of a photochromic protein or a variant or polypeptide fragmentthereof; L is an optional linker amino acid sequence; X is an amino acidsequence of a selected polypeptide of interest; A is an optionalN-terminal amino acid sequence; and B is an optional C-terminal aminoacid sequence. In fusion proteins comprising two photochromicpolypeptides, the photochromic polypeptides D₁ and D₂ can be the same ordifferent. For example, the fusion protein may comprise two Dronpa 145Npolypeptides, or two Dronpa 145K polypeptides, or a Dronpa 145Kpolypeptide and a Dronpa 145N polypeptide. Where more than one linker ispresent in the fusion, the linkers can also be the same or different.

Linker amino acid sequence(s) -L- will typically be short, e.g., 20 orfewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1). Examples include short peptide sequenceswhich facilitate cloning, poly-glycine linkers (Gly_(n) where n=2, 3, 4,5, 6, 7, 8, 9, 10 or more), histidine tags (His_(n) where n=3, 4, 5, 6,7, 8, 9, 10 or more), linkers composed of glycine and serine residues([Gly-Ser]_(n), [Gly-Gly-Ser-Gly]_(n) (SEQ ID NO:11),[Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:12), and [Ser-Ala-Gly-Gly]_(n) (SEQID NO:13), wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or more), GSAT, SEG, and Z-EGFR linkers. Linkers may include restrictionsites, which aid cloning and manipulation. Other suitable linker aminoacid sequences will be apparent to those skilled in the art. (See e.g.,Argos (1990) J. Mol. Biol. 211(4):943-958; Crasto et al. (2000) ProteinEng. 13:309-312; George et al. (2002) Protein Eng. 15:871-879; Arai etal. (2001) Protein Eng. 14:529-532; and the Registry of StandardBiological Parts (partsregistry.org/Protein_domains/Linker).

-A- is an optional N-terminal amino acid sequence. This will typicallybe short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1). Examplesinclude leader sequences to direct protein localization, or shortpeptide sequences or tag sequences, which facilitate cloning orpurification (e.g., a histidine tag His_(n) where n=3, 4, 5, 6, 7, 8, 9,10 or more). Other suitable N-terminal amino acid sequences will beapparent to those skilled in the art.

-B- is an optional C-terminal amino acid sequence. This will typicallybe short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1). Examplesinclude sequences to direct protein localization, short peptidesequences or tag sequences, which facilitate cloning or purification(e.g., His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more), or sequenceswhich enhance protein stability. Other suitable C-terminal amino acidsequences will be apparent to those skilled in the art.

In certain embodiments, tag sequences are located at the N-terminus orC-terminus of the fusion protein. Exemplary tags that can be used in thepractice of the invention include a His-tag, a Strep-tag, a TAP-tag, anS-tag, an SBP-tag, an Arg-tag, a calmodulin-binding peptide tag, acellulose-binding domain tag, a DsbA tag, a c-myc tag, a glutathioneS-transferase tag, a FLAG tag, a HAT-tag, a maltose-binding protein tag,a NusA tag, and a thioredoxin tag.

In certain embodiments, the fusion protein comprises a targetingsequence. Exemplary targeting sequences that can be used in the practiceof the invention include a secretory protein signal sequence, a membraneprotein signal sequence, a nuclear localization sequence, a nucleolarlocalization signal sequence, an endoplasmic reticulum localizationsequence, a peroxisome localization sequence, a mitochondriallocalization sequence, and a protein-protein interaction motif sequence.Examples of targeting sequences include those targeting the nucleus(e.g., KKKRK, SEQ ID NO:14), mitochondrion (e.g.,MLRTSSLFTRRVQPSLFRNILRLQST, SEQ ID NO:15), endoplasmic reticulum (e.g.,KDEL, SEQ ID NO:16), peroxisome (e.g., SKL), synapses (e.g., S/TDV orfusion to GAP 43, kinesin or tau), plasma membrane (e.g., CaaX (SEQ IDNO:17) where “a” is an aliphatic amino acid, CC, CXC, CCXX (SEQ IDNO:18) at C-terminus), or protein-protein interaction motifs (e.g., SH2,SH3, PDZ, WW, RGD, Src homology domain, DNA-binding domain, SLiMs).

In another aspect, the invention includes a method for controlling theactivity of a selected polypeptide of interest with light. The methodcomprises (i) preparing a fusion protein comprising a first photochromicpolypeptide connected to the N-terminus of the selected polypeptide ofinterest and a second photochromic polypeptide connected to theC-terminus of the selected polypeptide of interest; (ii) illuminatingthe fusion protein with light at a wavelength that inducesintramolecular dimerization of the first photochromic polypeptide andthe second photochromic polypeptide (e.g., about 405 nm for some fusionswith Dronpa 145N or 145K), such that the activity of the selectedpolypeptide of interest is inactivated. In certain embodiments, themethod further comprises illuminating the fusion protein with light thatinduces dissociation of the first photochromic polypeptide from thesecond photochromic polypeptide (e.g., about 480-500 nm for some fusionswith Dronpa 145N or 145K), such that the activity of the selectedpolypeptide is restored. Localization of the selected polypeptide aswell as inactivation of the selected polypeptide can be visualized bydetecting fluorescence of the fusion protein resulting fromintramolecular dimerization of the first photochromic polypeptide andthe second photochromic polypeptide in the fusion protein. Inactivationof the selected polypeptide can further be assessed by measuring theactivity of the selected polypeptide.

In another aspect, the invention includes a method for controlling thelocalization of a selected polypeptide of interest with light. Themethod comprises (i) preparing a first fusion protein comprising aphotochromic polypeptide connected to a targeting sequence; (ii)preparing a second fusion protein comprising a photochromic polypeptideconnected to the selected polypeptide of interest; (iii) introducing thefirst photochromic fusion and the second fusion protein into a cell,wherein the localization sequence targets the first fusion protein to aparticular subcellular location; (iv) and illuminating the fusionproteins with light at a wavelength that induces oligomerization of thephotochromic polypeptide in the first fusion protein with thephotochromic polypeptide in the second fusion protein (e.g., about 405nm for some fusions with Dronpa 145N or 145K), such that the selectedpolypeptide of interest accumulates at the subcellular location. Incertain embodiments, the method further comprises illuminating thefusion proteins with light that induces dissociation of the photochromicpolypeptides (e.g., about 480-500 nm for some fusions with Dronpa 145Nor 145K), such that the selected polypeptide of interest in the secondfusion protein is released from the subcellular location. Localizationof the selected polypeptide of interest can be visualized by detectingfluorescence of the fusion proteins resulting from the oligomerizationof the photochromic polypeptides.

In another aspect, the invention includes a method for controlling thelocalization of a selected polypeptide of interest with light. Themethod comprises: (i) preparing a fusion protein comprising aphotochromic polypeptide, a targeting sequence, and the selectedpolypeptide of interest; (ii) introducing the fusion protein into acell, wherein the localization sequence targets the fusion protein to aparticular subcellular location; and (iii) illuminating the fusionprotein with light at a wavelength that induces oligomerization of thephotochromic polypeptide in the fusion protein with photochromicpolypeptides in other fusion proteins (e.g., about 405 nm for somefusions with Dronpa 145N or 145K), said other fusion proteins comprisingthe selected polypeptide of interest, such that the selected polypeptideof interest accumulates at the subcellular location. In certainembodiments, the method further comprises illuminating the fusionprotein with light at a wavelength that induces dissociation of thephotochromic polypeptides (e.g., about 480-500 nm for some fusions withDronpa 145N or 145K), such that the selected polypeptide of interest inthe fusion protein is released from the subcellular location.Localization of the selected polypeptide of interest can be visualizedby detecting fluorescence of the fusion protein resulting from theoligomerization with photochromic polypeptides of the other fusionproteins.

In the practice of the invention, the fluorescence of fusion proteinscan be monitored by any suitable method. For example, fluorescence offusion proteins can be detected by a fluorimeter, a fluorescencemicroscope, a fluorescence microplate reader, a fluorometric imagingplate reader, or fluorescence-activated cell sorting.

B. Production of Fusion Proteins

Fusion proteins can be produced in any number of ways, all of which arewell known in the art. In one embodiment, the fusion proteins aregenerated using recombinant techniques. One of skill in the art canreadily determine nucleotide sequences that encode the desiredpolypeptides using standard methodology and the teachings herein.Oligonucleotide probes can be devised based on the known sequences andused to probe genomic or cDNA libraries. The sequences can then befurther isolated using standard techniques and, e.g., restrictionenzymes employed to truncate the gene at desired portions of thefull-length sequence. Similarly, sequences of interest can be isolateddirectly from cells and tissues containing the same, using knowntechniques, such as phenol extraction and the sequence furthermanipulated to produce the desired truncations. See, e.g., Sambrook etal., supra, for a description of techniques used to obtain and isolateDNA.

The sequences encoding polypeptides can also be produced synthetically,for example, based on the known sequences. The nucleotide sequence canbe designed with the appropriate codons for the particular amino acidsequence desired. The complete sequence is generally assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53.

Recombinant techniques are readily used to clone sequences encodingpolypeptides useful in the claimed fusion proteins that can then bemutagenized in vitro by the replacement of the appropriate base pair(s)to result in the codon for the desired amino acid. Such a change caninclude as little as one base pair, effecting a change in a single aminoacid, or can encompass several base pair changes. Alternatively, themutations can be effected using a mismatched primer that hybridizes tothe parent nucleotide sequence (generally cDNA corresponding to the RNAsequence), at a temperature below the melting temperature of themismatched duplex. The primer can be made specific by keeping primerlength and base composition within relatively narrow limits and bykeeping the mutant base centrally located. See, e.g., Innis et al,(1990) PCR Applications: Protocols for Functional Genomics; Zoller andSmith, Methods Enzymol. (1983) 100:468. Primer extension is effectedusing DNA polymerase, the product cloned and clones containing themutated DNA, derived by segregation of the primer extended strand,selected. Selection can be accomplished using the mutant primer as ahybridization probe. The technique is also applicable for generatingmultiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl.Acad. Sci USA (1982) 79:6409.

Once coding sequences have been isolated and/or synthesized, they can becloned into any suitable vector or replicon for expression. (See, also,Examples). As will be apparent from the teachings herein, a wide varietyof vectors encoding modified polypeptides can be generated by creatingexpression constructs which operably link, in various combinations,polynucleotides encoding polypeptides having deletions or mutationstherein.

Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

Insect cell expression systems, such as baculovirus systems, can also beused and are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalia, Invitrogen, San Diego Calif. (“MaxBac” kit).

Plant expression systems can also be used to produce the fusion proteinsdescribed herein. Generally, such systems use virus-based vectors totransfect plant cells with heterologous genes. For a description of suchsystems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; andHackland et al., Arch. Virol. (1994) 139:1-22.

Viral systems, such as a vaccinia based infection/transfection system,as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby etal., J. Gen. Virol. (1993) 74:1103-1113, will also find use with thepresent invention. In this system, cells are first transfected in vitrowith a vaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the DNA of interest, driven by a T7 promoter.The polymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA that is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation product(s).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. With the present invention, both thenaturally occurring signal peptides and heterologous sequences can beused. Leader sequences can be removed by the host in post-translationalprocessing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.Such sequences include, but are not limited to, the TPA leader, as wellas the honey bee mellitin signal sequence.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Such regulatory sequences are known to those of skillin the art, and examples include those which cause the expression of agene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector thatalready contains the control sequences and an appropriate restrictionsite.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. Mutants oranalogs may be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Vero293 cells, as well as others. Similarly, bacterial hosts such as E.coli, Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorphs, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa califormica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the fusionproteins of the present invention are produced by growing host cellstransformed by an expression vector described above under conditionswhereby the protein of interest is expressed. The selection of theappropriate growth conditions is within the skill of the art.

In one embodiment, the transformed cells secrete the polypeptide productinto the surrounding media. Certain regulatory sequences can be includedin the vector to enhance secretion of the protein product, for exampleusing a tissue plasminogen activator (TPA) leader sequence, aninterferon (γ or α) signal sequence or other signal peptide sequencesfrom known secretory proteins. The secreted polypeptide product can thenbe isolated by various techniques described herein, for example, usingstandard purification techniques such as but not limited to,hydroxyapatite resins, column chromatography, ion-exchangechromatography, size-exclusion chromatography, electrophoresis, HPLC,immunoadsorbent techniques, affinity chromatography,immunoprecipitation, and the like.

Alternatively, the transformed cells are disrupted, using chemical,physical or mechanical means, which lyse the cells yet keep therecombinant polypeptides substantially intact. Intracellular proteinscan also be obtained by removing components from the cell wall ormembrane, e.g., by the use of detergents or organic solvents, such thatleakage of the polypeptides occurs. Such methods are known to those ofskill in the art and are described in, e.g., Protein PurificationApplications: A Practical Approach, (Simon Roe, Ed., 2001).

For example, methods of disrupting cells for use with the presentinvention include but are not limited to: sonication or ultrasonication;agitation; liquid or solid extrusion; heat treatment; freeze-thaw;desiccation; explosive decompression; osmotic shock; treatment withlytic enzymes including proteases such as trypsin, neuraminidase andlysozyme; alkali treatment; and the use of detergents and solvents suchas bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. Theparticular technique used to disrupt the cells is largely a matter ofchoice and will depend on the cell type in which the polypeptide isexpressed, culture conditions and any pre-treatment used.

Following disruption of the cells, cellular debris is removed, generallyby centrifugation, and the intracellularly produced polypeptides arefurther purified, using standard purification techniques such as but notlimited to, column chromatography, ion-exchange chromatography,size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbenttechniques, affinity chromatography, immunoprecipitation, and the like.

For example, one method for obtaining the intracellular polypeptides ofthe present invention involves affinity purification, such as byimmunoaffinity chromatography using antibodies (e.g., previouslygenerated antibodies), or by lectin affinity chromatography.Particularly preferred lectin resins are those that recognize mannosemoieties such as but not limited to resins derived from Galanthusnivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentillectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissuspseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA).The choice of a suitable affinity resin is within the skill in the art.After affinity purification, the polypeptides can be further purifiedusing conventional techniques well known in the art, such as by any ofthe techniques described above.

Polypeptides can be conveniently synthesized chemically, for example byany of several techniques that are known to those skilled in the peptideart. In general, these methods employ the sequential addition of one ormore amino acids to a growing peptide chain. Normally, either the aminoor carboxyl group of the first amino acid is protected by a suitableprotecting group. The protected or derivatized amino acid can then beeither attached to an inert solid support or utilized in solution byadding the next amino acid in the sequence having the complementary(amino or carboxyl) group suitably protected, under conditions thatallow for the formation of an amide linkage. The protecting group isthen removed from the newly added amino acid residue and the next aminoacid (suitably protected) is then added, and so forth. After the desiredamino acids have been linked in the proper sequence, any remainingprotecting groups (and any solid support, if solid phase synthesistechniques are used) are removed sequentially or concurrently, to renderthe final polypeptide. By simple modification of this general procedure,it is possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M.Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo., Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, ThePeptides: Analysis, Synthesis, Biology, editors E. Gross and J.Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, forsolid phase peptide synthesis techniques; and M. Bodansky, Principles ofPeptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J.Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1,for classical solution synthesis. These methods are typically used forrelatively small polypeptides, i.e., up to about 50-100 amino acids inlength, but are also applicable to larger polypeptides.

Typical protecting groups include t-butyloxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl,acetyl, o-nitrophenylsulfonyl and the like.

Typical solid supports are cross-linked polymeric supports. These caninclude divinylbenzene cross-linked-styrene-based polymers, for example,divinylbenzene-hydroxymethylstyrene copolymers,divinylbenzene-chloromethylstyrene copolymers anddivinylbenzene-benzhydrylaminopolystyrene copolymers.

Polypeptide analogs can also be chemically prepared by other methodssuch as by the method of simultaneous multiple peptide synthesis. See,e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Pat.No. 4,631,211.

C. Kits

Fusion proteins or nucleic acids encoding them can be provided in kitswith suitable instructions and other necessary reagents for preparing orusing the fusion proteins, as described above. The kit may contain inseparate containers fusion proteins, or recombinant constructs forproducing fusion proteins, and/or cells (either already transfected orseparate). Additionally, instructions (e.g., written, tape, VCR, CD-ROM,DVD, etc.) for using the fusion proteins may be included in the kit. Thekit may also contain other packaged reagents and materials (e.g.,transfection reagents, buffers, media, and the like).

D. Applications

The fusion proteins of the invention provide useful tools for spatiallyand temporally controlling protein activity with light and will findnumerous applications in basic research and development. In particular,fusion proteins can be designed to block or induce activities ofproteins of interest, control their interactions with othermacromolecules, or direct their subcellular localization. Because fusionproteins can potentially be used to control diverse cellular processeswith light, they will be especially useful in the study of proteinfunction in physiological processes and disease mechanisms.

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Optical Control of Protein Activity by Fusion to FluorescentProtein Domains Introduction

Here, we describe the discovery of an engineered protein interactionthat is controlled by cyan light and requires no cofactors. We use thislight-controlled association to develop a simple generalizable designfor light-inducible proteins. We created a fluorescent light-inducibleprotein design in which Dronpa domains are fused to both termini of anenzyme domain. In the dark, the Dronpa domains associate and cage theprotein, but light induces Dronpa dissociation and activates theprotein. This method enabled optical control over guanine nucleotideexchange factor (GEF) and protease domains without extensive screening.Our findings extend the applications of fluorescent proteins fromexclusively sensing functions to also encompass optogenetic control.

Dronpa is a monomeric fluorescent protein (FP) derived from thetetrameric parent protein, 22G, isolated from a Pectiniidae genus coral(Ando et al. (2004) Science 306:1370-1373). Fluorescence of Dronpaswitches off under cyan light (˜500 nm) and switches on under violetlight (˜400 nm) (Ando et al., supra). With off-photoswitching, β strand7 near the chromophore becomes flexible (Mizuno et al. (2008) Proc.Natl. Acad. Sci. U.S.A. 105:9227-9232); this strand forms part of thecross-dimer interface in the tetrameric parent (Mizuno et al. (2008),supra). A Dronpa mutant with Lys¹⁴⁵ on β strand 7 changed to Asn(Dronpa145N) is tetrameric at low micromolar concentrations, butdilution promotes monomerization and facilitates off-photoswitching(Mizuno et al. (2010) Photochem. Photobiol. Sci. 9: 239-248). Thissuggests that multimerization inhibits conformation changes associatedwith off-photoswitching. We hypothesized, conversely, that conformationchanges occurring during off-photoswitching might promotemonomerization, whereas on-photoswitching might promote multimerization(FIG. 1A).

Materials and Methods

DNA Construction

pcDNA3-mNeptune1-fascin was a gift of Michael W. Davidson (Florida StateUniversity, Tallahassee). tdTomato and mCherry plasmids were gifts ofNathan Shaner and Roger Y. Tsien (UCSD). Dronpa145K and Dronpa145N weresynthesized by polymerase chain reaction (PCR) of overlappingoligonucleotides and cloned into pNCS, a constitutive bacterialexpression vector with a six-consecutive-histidine tag at its N-terminusfor purification and BamHI and EcoRI sites for insert cloning (Müller etal. (2008) ChemBioChem 9:2029-2038; herein incorporated by reference). Aconstruct encoding amino acids 1234-1428 of the human intersectin DHdomain (Entrez Gene ID 6453) was synthesized by PCR from overlappingoligonucleotides and cloned into the mammalian expression vector pcDNA3(Invitrogen). Plasmids containing HCV protease and substrate sequencesand mRuby2 were previously described (Faix et al. (2009) Int. J.Biochem. Cell Biol. 41:1656-1664; Lam et al. (2012) Nat. Methods9:1005-1012; herein incorporated by reference).

In addition to pNCS-Dronpa145K and pNCS-Dronpa145N described above, tocreate other bacterial expression constructs for native polyacrylamidegel electrophoresis, mRuby2, tdTomato, and DsRed2 open reading frames(ORFs) were amplified from pBAD-tdTomato, pcDNA3-mRuby2, and pDsRed2-N1(Clontech), respectively, and cloned into pNCS.pNCS-tdDronpa145K-Dronpa145N was created by recombination of aPCR-amplified Dronpa145K ORF with BamHI-digested pNCS-Dronpa145N usingthe In-Fusion recombinase (Clontech).

To create mammalian expression plasmids for fusions of Dronpa andintersectin domains, PCR fragments encoding Dronpa and intersectin DHdomains and the Kras4B CAAX sequence (KMSKDGKKKKKKSKTKCVIM, SEQ IDNO:21) were amplified from the above plasmids or from overlapping oligos(for the CAAX sequence), then assembled in a second PCR reaction andcloned into pcDNA3. To create plasmids coexpressingmCherry-substrate-CAAX and fusions of Dronpa and HCV NS4A/NS3 protease,the lentiviral vector pLL3.7 (Addgene) was first modified to reduce itssize by replacing the untranslated sequence between PvuII and BspEIsites upstream of the 3′ long terminal repeat with a more compactsequence containing only the polypyrimidine tract and integrase att sitenecessary for reverse transcription and integration, creating pLL3.7m.Then a fusion of mCherry, the NS4A/NS4B substrate sequence, and CAAX wasassembled by overlapping PCR and inserted between NheI and EcoRV sitesdownstream of the CMV promoter by ligation, creatingpLL3.7m-mCherry-substrate-CAAX. Finally, PCR fragments encoding aminimal CMV promoter, Dronpa and HCV NS4A/NS3 protease domains, and SV40polyadenylation signals from pcDNA3 were assembled and inserted betweenthe NotI and XbaI sites of pLL3.7m-mCherry-substrate-CAAX by theIn-Fusion recombinase. This created an expression cassette adjacent toand in the opposite transcriptional direction from the original CMVpromoter.

In Vitro Protein Characterization and Photoswitching

Bacterial expression plasmids for Dronpa145K, Dronpa145N,tdDronpa145K-Dronpa145N, mRuby, tdTomato, and dsRed2 were transformedinto chemically competent Escherichia coli strain DH5α for expression. Asingle colony was inoculated into 100 ml of Luria-Bertani (LB) brothcontaining 50 μg ml⁻¹ ampicillin and incubated overnight at 37° C. Thecultures were further incubated at room temperature for another 24hours, then fluorescent protein purification from bacterial lysates wasperformed by polyhistidine affinity purification as previously described(Müller et al., supra). Protein concentrations were estimated byabsorbance spectrophotometry and purity was verified by SDS-PAGE. Forcharacterization of baseline oligomerization state, 5 μL each of 100 μM,20 μM, or 10 μM of Dronpa145K or Dronpa145N were run on a 4-16% Bis-Trisnative PAGE gel (Invitrogen) with dark cathode buffer alongside 5 μLeach of 20 μM mRuby2, tdTomato, and dsRed2 as size controls.

For in vitro photoswitching, purified Dronpa145N and tandem dimerDronpa145K-Dronpa145N proteins were diluted to 100 μM. Proteins in a0.2-mL PCR tube were switched off by placement between two cyan LEDsmounted 1 inch apart for 30 minutes (505/30 nm, 170 mW, Thorlab). Thefluorescence recovery was conducted by illumination with two similarlymounted UV LEDs for 30 seconds (405/20 nm, 470 mW, Thorlab), followed byincubation at room temperature for 30 minutes. The switching efficiencywas estimated by measuring the fluorescence of 1-μL protein aliquotsusing a Safire 2 monochromator-based fluorescence spectrophotometer(TECAN). In parallel, 2.5 μL (Dronpa145N) or 5 μL(tdDronpa145K-Dronpa145N) of the protein in each condition were loadedon a 4-16% Bis-Tris native PAGE gel (Invitrogen) with dark cathodebuffer. 5 μL each of 20 μM mRuby, tdTomato, and dsRed2 were loaded assize controls.

Cell Culture and Transfection

Cells were maintained in high glucose Dulbecco's Modified Eagle Medium(DMEM, HyClone) supplemented with 10% fetal bovine serum (FBS,Invitrogen) and 2 mM glutamine (Sigma) at 37° C. in air with 5% carbondioxide. Hela cells were transfected at 75-90% confluency withLipofectamine 2000 (Invitrogen) in 33-mm coverglass-bottom dishes (InVitro Scientific). Transfections were carried out according tomanufacturer's instructions, except that amounts of DNA and transfectionreagent were halved to reduce cell toxicity. NIH 3T3 cells (5-7×10⁴)were plated directly in a transfection solution containing DNA plasmidsand Lipofectamine 2000 in 33-mm coverglass-bottom dishes. Amounts of DNAand transfection reagent were reduced to ⅕ of themanufacture-recommended amount for a 33 mm culture. For both HeLa andNIH 3T3 cells, the medium was refreshed 4-6 hours after transfection.HEK293 cells were grown in 8 well-chambered coverglass (Nunc) andtransfected at 75-90% confluency using Lipofectamine LTX (Invitrogen)according to the manufacturer's instructions.

Membrane Translocation and Protein Uncaging

In the translocation assay, Hela cells were imaged in PBS at roomtemperature 12-36 hours after transfection. Imaging was performed with aC-Apochromat 40×1.2 numerical aperture (NA) water-immersion objective ona Zeiss Axiovert 200 M with a Ludl excitation filter controlled by aLudl MAC 5000 controller, using a Hamamatsu Orca ER Firewire camera. Allinstruments were controlled by a 2.5 Ghz MacBook Pro computer runningMicro-manager 1.4 software in Mac OS 10.6.8. Illumination was providedby a 120-W metal-halide light source (Exfo) passed through a 1-m liquidlight guide with a 3 mm core. Dronpa was imaged with a 10% neutraldensity filter, a 485/30-nm excitation filter, a 505-nm dichroic mirror,and a 525/40 nm emission filter. Neptune was imaged with a 10% neutraldensity filter, a 560/40 nm excitation filter, a 585 nm dichroic mirror,and a 630/75 emission filter. Dronpa was photoswitched off byillumination using the Dronpa channel excitation and dichroic filtersand no neutral density filter for the indicated times. The lightintensity was measured to be 4.7 W cm⁻². Images were acquired within 2minutes after photoswitching to report Dronpa photoswitching andmNeptune movements. Light passed through a 10% neutral density filterand a 405/20 nm filter and a 440 nm dichroic mirror was used to recoverDronpa fluorescence. Images were acquired immediately to report Dronparecovery and 5 minutes later to report the mNeptune movements.

For intersectin experiments, NIH 3T3 cells were incubated in serum-freeDMEM media for 5-9 hours beginning 32-48 hours after transfection. Cellswere then imaged in HBSS at room temperature as described above. Dronpawas photoswitched as described above. Cells were imaged at intervals5-10 minutes apart for up to 1 hour.

For protease experiments, HEK293 cells were imaged 16 hours aftertransfection in HBSS with 2% B27 (Invitrogen), 1 mM sodium pyruvate, and10 mM HEPES pH 7.2 in a TC CU109 chamber (Chamide) heated to 37° C.Imaging was done with an Olympus 40× 1.15 NA water immersion objectiveon Olympus IX80 with a Ludl excitation filter controlled by a Ludl MAC5000 controller, using a Hamamatsu Orca ER Firewire camera. Allinstruments were controlled by a 3 GHz Dell Optiplex 755SFF computerrunning Micro-manager 1.4 software in Windows 7. Illumination wasprovided by a 120-W metal-halide light source (Exfo) passed through a1-m liquid light guide with a 3 mm core. Dronpa was imaged with a 485/22nm excitation filter, 510 nm dichroic mirror, and 540/40 nm emissionfilter. The mCherry was imaged with a 545/30 nm excitation filter, 570nm dichroic mirror, and 605/50 nm emission filter. Dronpa wasphotoswitched off by 10 seconds of illumination using the Dronpa channelexcitation and dichroic filters and no neutral density filter. Imageswere acquired at 10 minutes, 30 minutes and 60 minutes afterphotoswitching.

Statistical Analysis

To determine statistical significance of light-dependent filopodiainduction, the Pearson chi-squared test was performed on thedistributions of the two observation outcomes of filopodia induction orno filopodia induction between two treatment conditions of illuminationor no illumination. The null hypothesis was that filopodia induction isindependent of treatment condition. A responding cell was defined as acell with at least one new filopodium per polygonal side.

Results

To determine if light could control Dronpa145N multimerization, weperformed native polyacrylamide gel electrophoresis (PAGE). Dronpa145Nwas tetrameric at concentrations from 10 to 100 μM in the initial brightstate, whereas wild-type Dronpa (Dronpa145K for clarity; K, Lys) wasmonomeric (FIG. 5A). Cyan illumination of 100 μM Dronpa145N induced ashift from a cyan-absorbing to a violet-absorbing species (FIG. 1C) anda loss of green fluorescence (FIG. 5B), as previously described (Ando etal. (2004) Science 306: 1370-1373). Simultaneously, Dronpa145Nredistributed from tetrameric toward monomeric species (FIG. 1B, lane2), implying that off-photoswitched Dronpa145N has a dissociationconstant exceeding 100 μM. Violet light restored cyan absorbance (FIG.1C) and green fluorescence (FIG. 5B) and also induced retetramerization(FIG. 1B, lane 3), indicating that monomerization was not due toirreversible protein damage. These results show that Dronpa145Ninteractions can be controlled by light.

A dimer-to-monomer conversion might be more easily harnessed to controlprotein activity than a tetramer-to-monomer conversion. Given the lackof multimerization of Dronpa 145K, we explored whether oligomerizationof Dronpa145K and Dronpa145N could be limited to dimerization. Toachieve high effective concentrations of Dronpa145K and Dronpa145Nwithout driving Dronpa145N tetramerization, we fused Dronpa145K intandem to Dronpa145N via a linker (K-N tandem dimer) (FIG. 1D). Theeffective concentration of one domain relative to another on the samepolypeptide has been estimated at ˜70 μM (Müller et al., supra). The K-Nconstruct migrated in native PAGE primarily as expected for a tandemdimer (FIG. 1E). If the Dronpa domains were engaged in a light-sensitiveintramolecular interaction, illumination should induce dissociation,resulting in a more elongated faster-migrating conformation. Indeed, thetandem dimer migrated faster after cyan illumination, and this processwas reversed after violet light-induced recovery (FIG. 1E). Expectedtransitions between cyan- and violet-absorbing forms were again observed(FIG. 1F and FIG. 5C). Thus, the K-N tandem dimer undergoes reversiblelight-induced conformational changes consistent with dissociation andreassociation of Dronpa domains.

To determine whether light-induced Dronpa145N dissociation could occurin mammalian cells, we created two fusions: N-CAAX, a fusion ofDronpa145N to the membrane-anchoring K-Ras C-terminal farnesylationmotif (CAAX box), and mNeptune-N, a fusion of the far-red FP mNeptune toDronpa145N (FIG. 2A) (Lin et al. (2009) Chem. Biol. 16:1169-1179). Upon10-fold relative overexpression of N-CAAX to insure an excess ofmembrane-localized Dronpa, some mNeptune-N was membrane-bound throughDronpa145N oligomerization (FIGS. 2C and 2D). Cyan light switched offDronpa fluorescence (FIG. 2B) and resulted in the release of mNeptunefrom the membrane (FIGS. 2C and 2D). Release required prolongedexposures (2 minutes, metal halide lamp at 100% neutral density througha 40×1.2-numerical aperture lens) and was only partial, but neverthelessindicated that light could induce Dronpa domain dissociation in cells.

To find conditions for Dronpa domain dissociation that require lesslight, we explored Dronpa145K-Dronpa145N heterodimerization (FIG. 2E).Dronpa145K-CAAX (K-CAAX) was able to recruit mNeptune-N to the membrane(FIG. 2G). Off-photoswitching of membrane fluorescence was faster thanwith N-CAAX (FIG. 2F), and release of mNeptune required only 20 secondsof illumination (FIGS. 2G and 2H). On-photoswitching of Dronpa by violetlight induced membrane re-localization of mNeptune-N (FIGS. 2G and 2H).Reversing the positions of Dronpa domains by expressing N-CAAX andmNeptune-Dronpa145K (mNeptune-K) did not result in a membrane mNeptunesignal (FIG. 6A), perhaps because tetramerization between concentratedN-CAAX molecules outcompeted weaker heterodimerization with mNeptune-K.Use of only monomeric Dronpa domains (K-CAAX and mNeptune-K) alsoresulted in no membrane mNeptune (FIG. 6B), as expected.

We hypothesized that we could use Dronpa to build light-controllablesingle chain proteins. Specifically, we hypothesized that proteinfunctions could be blocked by fusing Dronpa domains to the aminoterminus (NT) and the carboxyl terminus (CT) (FIG. 7A). Binding of thetwo Dronpa domains would “cage” the protein in an inactive state bymasking surfaces required for binding interaction partners orsubstrates, similarly to auto-inhibition of many kinases (Leonard et al.(2007) Cell 129:1037-1038), transcription factors (Pufall et al. (2002)Annu Rev. Cell Dev. Biol. 18:421-462), and guanine nucleotide exchangefactors (GEFs) for monomeric guanosine triphosphatases (GTPases) (Yu etal. (2010) Cell 140:246-256). Protein function could then be induced bylight-mediated dissociation of the Dronpa domains (FIG. 7A).

We first controlled the Cdc42 GEF intersectin, which can be inactivatedby terminal circularization (Yeh et al. (2007) Nature 447:596-600). Wefused Dronpa145K or Dronpa145N at the NT of the intersectin Dbl homology(DH) domain and Dronpa145N at the CT followed by the CAAX sequence,creating K-I-N-CAAX and N-I-N-CAAX (FIG. 7B). As catalytically activecontrols, we fused Dronpa145K to either side of intersectin (K-I-CAAXand I-K-CAAX) (FIG. 7B). We coexpressed these constructs in fibroblastswith a mNeptune-fascin reporter to mark filopodia and lamellipodia(Adams (2004) Curr. Opin. Cell Biol. 16:590-596). I-K-CAAX or K-I-CAAXrobustly induced filopodia and lamellipodia (FIGS. 7C and 7D), asexpected for Cdc42 activation, which induces filopodia directly andlamellipodia directly via the formin-family protein FMNL2 and indirectlyvia Rac (Block et al. (2012) Curr. Biol. 22:1005-1012; Nishimura et al.(2005) Nat. Cell Biol. 7:270-277). Cells expressing N-I-N-CAAX andK-I-N-CAAX produced filopodia or lamellipodia at much lower frequenciesthan I-K-CAAX or K-I-CAAX (FIGS. 7C and 7D). These experiments wereperformed by transient transfection, which results in variableexpression levels. When designated as low, medium, or high expressers byDronpa fluorescence (FIGS. 8A and 8C), low expressers, which includedthe majority of cells, exhibited basal filopodia or lamellipodiainfrequently (0% for N-I-N-CAAX and 8% for K-I-N-CAAX) (FIGS. 8B and8D). Thus, fusion of flanking Dronpa domains cages intersectin activityeffectively as long as higher expression levels are avoided, similarlyto tophototropin-based photoactivable Rac (PA-Rac; Wu et al. (2011)Methods Enzymol. 497:393-407).

We next asked whether caged intersectins could mediate filopodia orlamellipodia induction by light (FIGS. 3A and 3E). Illumination with490/20-nm light for 30 seconds switched off more than 50% of thefluorescence in both N-I-N-CAAX- and K-I-N-CAAX-transfected fibroblasts(FIGS. 3B and 3F). This light dose induced abundant filopodia formationwithin 30 minutes in 78% of cells expressing N-I-N-CAAX (FIG. 3C andFIG. 9D). This response was light-dependent, as only 10% of cellsexpressing N-I-N-CAAX formed filopodia in the same time interval withoutillumination (P<0.0001 by Pearson χ² test) (FIGS. 9A and 9D). Cellscontinued to exhibit filopodial mobility throughout 1 hour ofobservation and did not show blebbing that might indicate phototoxicity.Similarly, 90% of cells expressing K-I-N-CAAX formed abundant filopodiawithin 30 minutes after illumination (FIG. 3G, FIG. 9D), compared with25% not exposed to light (P<0.0001 by Pearson x² test) (FIGS. 9B and9D). Illumination of K-CAAX-expressing cells did not induce filopodia(FIGS. 9C and 9D), confirming that the effect is not due to light alone.These results demonstrate that a protein caged by Dronpa fusion can beuncaged by light.

We investigated whether caged intersectin constructs could controlfilopodia formation with spatial or temporal specificity. First, weperformed local illumination (490/20-nm light for 30 seconds) toportions of cells expressing N-I-N-CAAX or K-IN-CAAX and observed thatfilopodia appeared specifically in the illuminated regions (FIGS. 3D and3H). We next tested whether light could induce filopodia in differentlocations at different times in one cell. We applied a 30-seconduncaging pulse of cyan light at one subcellular region, a 3-secondglobal recaging pulse of violet light, and finally another 30-seconduncaging pulse at a different subcellular region. After the firstuncaging pulse, filopodia appeared in the first region, whereas afterthe global recaging and second uncaging pulse, filopodia appeared in thesecond region simultaneous with retraction in the first region (FIG.10).

Whether Cdc42 activation can lengthen existing filopodia has beenunclear, as Cdc42 effectors that promote filopodia extension rather thaninitiation have not been found. Rapid induction of intersectin activityby light allowed us to address this question. We observed thatphotouncaging of intersectin caused lengthening of many preexistingfilopodia (FIG. 11). This suggests that models in which Cdc42 governsonly filopodia initiation are incomplete (Faix et al. (2009) Int. J.Biochem. Cell Biol. 41:1656-1664) and that effectors may exist thatpromote filopodia extension analogous to how FMNL2 promotes lamellipodiaextension downstream of Cdc42 (Block et al. (2012) Curr. Biol.22:1005-1112).

An attractive feature of our design is potential generalizability. Othermethods for optical control of single polypeptides, such as fusion toxanthopsin or phototropin, require extensive screening to achievecoupling of light-induced conformational changes to protein activationand, thus, have been applied to only a few targets (Fan et al. (2011)Biochemistry 50:1226-1237; Wu et al. (2009) Nature 461:104-108;Strickland et al. (2010) Nat. Methods 7:623-626). Our caged proteindesign does not require precise linkages; therefore, it should be moreeasily generalizable. Proteases are a class of enzymes for which lightactivation has not yet been achieved. Unlike GTPases or kinases,proteases are not naturally regulated by membrane recruitment,preventing the use of reversible membrane targeting methods to controlthem. Hence, we investigated whether we could create a light-inducibleprotease by fusion to Dronpa domains. We chose to regulate the hepatitisC virus (HCV) NS3-4A protease because its high sequence specificity andlack of overt toxicity allows assessment of function in mammalian cells(Lin et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105:7744-7749).Furthermore, it is composed predominantly of β strands and loops (Romanoet al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:20986-20991), providinga structural contrast to the completely α-helical DH domain (Snyder etal. (2002) Nat. Struct. Biol. 9:468-475).

We constructed a Dronpa145N-protease-Dronpa145N fusion (N-protease-N)and, as a protease reporter, a fusion of mCherry, the NS4A/NS4B cleavagesite of HCV polypeptide, and the CAAX-box farnesylation signal(mCherry-substrate-CAAX) (FIG. 4A). We expected that mCherryfluorescence would be released from the membrane into the cytosol byprotease activity. Indeed, mCherry signal was membrane-bound in cellsexpressing mCherry-substrate-CAAX alone and cytoplasmic in cellscoexpressing a positive control Dronpa145K-protease (FIG. 4B). We thenused mCherry-substrate-CAAX to report light induction of N-protease-N.After off-switching of Dronpa fluorescence, cells showed an increase incytosolic mCherry within 10 minutes, which continued to increase over 60minutes (FIG. 4C). This response required illumination (FIG. 12A) andprotease (FIG. 12B). Thus, the caged protein design can be used tocontrol an enzyme domain that is not easily regulated by relocalizationwithin the cell.

Since their discovery, FPs have seen widespread use exclusively assensing tools. We discovered that photochromic FPs can have dualidentities as optical sensors and light-controlled actuators. We havetranslated this discovery into a simple design for opticallycontrollable proteins, which we propose to call FLIPs, for fluorescentlight-inducible proteins. FLIPs also serve as their own reporters, asthe photochromic FP domains report both protein localization andactivity state. Thus, our results place photochromic FPs in a distinctcentral location in the optogenetic toolbox, integrating both sensingand controlling functions in a single protein class.

While the preferred embodiments of the invention have been illustratedand described, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for controlling the activity of aselected polypeptide of interest with light, the method comprising: a)preparing a fusion protein comprising at least two photochromicpolypeptides connected to a selected polypeptide of interest, wherein afirst photochromic polypeptide is connected to the N-terminus of theselected polypeptide of interest and a second photochromic polypeptideis connected to the C-terminus of the selected polypeptide of interest,wherein the first photochromic polypeptide and the second photochromicpolypeptide are capable of associating with each other, wherein theoligomerization state of the first photochromic polypeptide and thesecond photochromic polypeptide is controllable with light, wherein atleast one photochromic polypeptide is an rsTagRFP polypeptide or anmApple polypeptide; and b) illuminating the fusion protein with light ata wavelength that induces intramolecular dimerization of the firstphotochromic polypeptide and the second photochromic polypeptide, suchthat the activity of the selected polypeptide of interest isinactivated.
 2. The method of claim 1, further comprising illuminatingthe fusion protein with light at a wavelength that induces dissociationof the first photochromic polypeptide from the second photochromicpolypeptide, such that the activity of the selected polypeptide isrestored.
 3. The method of claim 1, further comprising visualizing thelocalization of the selected polypeptide by detecting fluorescence ofthe fusion protein resulting from the dimerization of the firstphotochromic polypeptide and the second photochromic polypeptide.
 4. Themethod of claim 1, further comprising detecting inactivation of theselected polypeptide by measuring fluorescence from dimerization of thefirst photochromic polypeptide and the second photochromic polypeptide.5. The method of claim 1, further comprising detecting inactivation ofthe selected polypeptide by measuring the activity of the selectedpolypeptide.
 6. The method of claim 1, wherein fluorescence of thefusion protein is detected by a fluorimeter, a fluorescence microscope,a fluorescence microplate reader, a fluorometric imaging plate reader,or fluorescence-activated cell sorting.
 7. The method of claim 1,wherein the first photochromic polypeptide or the second photochromicpolypeptide comprises: a) an amino acid sequence selected from the groupconsisting of SEQ ID NO:7 and SEQ ID NO:9; or b) an amino acid sequencehaving at least 95% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:7 and SEQ ID NO:9, wherein the polypeptidehas fluorescence and oligomerization characteristics.
 8. A method forcontrolling the localization of a selected polypeptide of interest withlight, the method comprising: a) preparing a first fusion proteincomprising a photochromic polypeptide connected to a targeting sequence;b) preparing a second fusion protein comprising a photochromicpolypeptide connected to the selected polypeptide of interest; c)introducing the first fusion protein and the second fusion protein intoa cell, wherein the localization sequence targets the first fusionprotein to a particular subcellular location; d) illuminating the fusionproteins with light at a wavelength that induces oligomerization of thephotochromic polypeptide in the first fusion protein with thephotochromic polypeptide in the second fusion protein, such that theselected polypeptide accumulates at the subcellular location.
 9. Themethod of claim 8, further comprising illuminating the fusion proteinswith light at a wavelength that induces dissociation of the photochromicpolypeptides, such that the selected polypeptide in the second fusionprotein is released from the subcellular location.
 10. The method ofclaim 8, further comprising visualizing the localization of the selectedpolypeptide by detecting fluorescence of the fusion proteins resultingfrom the oligomerization of the photochromic polypeptides.
 11. Themethod of claim 8, wherein the targeting sequence is selected from thegroup consisting of a secretory protein signal sequence, a membraneprotein signal sequence, a nuclear localization sequence, a nucleolarlocalization signal sequence, an endoplasmic reticulum localizationsequence, a peroxisome localization sequence, a mitochondriallocalization sequence, and a protein binding motif sequence.
 12. Themethod of claim 8, wherein fluorescence of the fusion proteins aredetected by a fluorometer, a fluorescence microscope, a fluorescencemicroplate reader, a fluorometric imaging plate reader, orfluorescence-activated cell sorting.
 13. The method of claim 8, whereinthe photochromic polypeptide in the first fusion protein and thephotochromic polypeptide in the second fusion protein are selected fromthe group consisting of a Dronpa polypeptide, a Padron polypeptide, anrsTagRFP polypeptide, and an mApple polypeptide.
 14. The method of claim13, wherein the photochromic polypeptide of the first fusion protein orthe second fusion protein comprises: a) an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9; or b) anamino acid sequence having at least 95% identity to an amino acidsequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7,and
 9. 15. The method of claim 13, wherein the photochromic polypeptidein the first fusion protein is a Dronpa-145N polypeptide or aPadron-145N polypeptide.
 16. The method of claim 13, wherein thephotochromic polypeptide in the first fusion protein is a Dronpa-145Kpolypeptide.
 17. The method of claim 13, wherein the photochromicpolypeptide in the second fusion protein is a Dronpa-145N polypeptide ora Padron-145N polypeptide.
 18. The method of claim 13, wherein thephotochromic polypeptide in the second fusion protein is a Dronpa-145Kpolypeptide.
 19. A method for controlling the localization of a selectedpolypeptide of interest with light, the method comprising: a) preparinga fusion protein comprising a photochromic polypeptide, a targetingsequence, and the selected polypeptide of interest; b) introducing thefusion protein into a cell, wherein the localization sequence targetsthe fusion protein to a particular subcellular location; c) illuminatingthe fusion protein with light at a wavelength that inducesoligomerization of the photochromic polypeptide in the fusion proteinwith photochromic polypeptides in other fusion proteins of the sametype, said fusion proteins comprising the selected polypeptide ofinterest, such that the selected polypeptide of interest accumulates atthe subcellular location.
 20. The method of claim 19, further comprisingilluminating the fusion protein with light at a wavelength that inducesdissociation of the photochromic polypeptides, such that the selectedpolypeptide of interest in the fusion protein is released from thesubcellular location.
 21. The method of claim 19, further comprisingvisualizing the localization of the selected polypeptide of interest bydetecting fluorescence of the fusion protein resulting from theoligomerization with the photochromic polypeptides of the other fusionproteins.
 22. The method of claim 19, wherein the targeting sequence isselected from the group consisting of a secretory protein signalsequence, a membrane protein signal sequence, a nuclear localizationsequence, a nucleolar localization signal sequence, an endoplasmicreticulum localization sequence, a peroxisome localization sequence, amitochondrial localization sequence, and a protein binding motifsequence.
 23. The method of claim 19, wherein the photochromicpolypeptide of the fusion protein comprises an amino acid sequenceselected from the group consisting of a Dronpa polypeptide, a Padronpolypeptide, an rsTagRFP polypeptide, and an mApple polypeptide.
 24. Themethod of claim 23, wherein the photochromic polypeptide of the fusionprotein comprises: a) an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1, 3, 5, 7, and 9; or b) an amino acid sequencehaving at least 95% identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOS:1, 3, 5, 7, and 9, wherein thephotochromic polypeptide has fluorescence and oligomerizationcharacteristics.
 25. The method of claim 23, wherein the photochromicpolypeptide of the fusion protein is a Dronpa-145N polypeptide or aPadron-145N polypeptide.
 26. The method of claim 23, wherein thephotochromic polypeptide of the fusion protein is an mApple-162H-164Apolypeptide.